Antimicrobial resistance profiles of Escherichia coli isolated from clinical and environmental samples: findings and implications

Abstract Background The overuse and misuse of antimicrobials has worsened the problem of antimicrobial resistance (AMR) globally. This study investigated the AMR profiles of Escherichia coli isolated from clinical and environmental samples in Lusaka, Zambia. Methods This was a cross-sectional study conducted from February 2023 to June 2023 using 450 samples. VITEK® 2 Compact was used to identify E. coli and perform antimicrobial susceptibility testing. Data analysis was done using WHONET 2022 and SPSS version 25.0. Results Of the 450 samples, 66.7% (n = 300) were clinical samples, whereas 33.3% (n = 150) were environmental samples. Overall, 47.8% (n = 215) (37.8% clinical and 10% environmental) tested positive for E. coli. Of the 215 E. coli isolates, 66.5% were MDR and 42.8% were ESBL-producers. Most isolates were resistant to ampicillin (81.4%), sulfamethoxazole/trimethoprim (70.7%), ciprofloxacin (67.9%), levofloxacin (64.6%), ceftriaxone (62.3%) and cefuroxime (62%). Intriguingly, E. coli isolates were highly susceptible to amikacin (100%), imipenem (99.5%), nitrofurantoin (89.3%), ceftolozane/tazobactam (82%) and gentamicin (72.1%). Conclusions This study found a high resistance of E. coli to some antibiotics that are commonly used in humans. The isolation of MDR and ESBL-producing E. coli is a public health concern and requires urgent action. Therefore, there is a need to instigate and strengthen interventional strategies including antimicrobial stewardship programmes to combat AMR in Zambia.


Introduction
1][22][23] E. coli has developed resistance to many antimicrobials using the natural resistance process. 24Additionally, the overuse and misuse of antimicrobials have led to the high level of resistance to these drugs in E. coli.This high resistance of E. coli to antimicrobials has been reported in human health, [25][26][27][28] animal health [29][30][31][32][33] and the environment. 25,34The occurrence of MDR

Sample size estimation and sampling
The sample size was estimated using the Cochran formula assuming a prevalence of E. coli of 64.5% as determined from a previous study in Zambia. 25Assuming an error rate of 5% and a non-response of 20% we determined the minimum sample size required to be 423.The sampling was conducted consecutively using convenience sampling.Only samples stored at room temperature for no more than 2 h were considered for the investigation.Samples that had incomplete demographic information were excluded from the study.

Data collection
The data collected from clinical samples included the date and time of sample collection, the type of sample collected, an identifying code, and the age and gender of the patients.Environmental samples provided additional information on the source, type and sampled area.

Quality control
The quality control (QC) of all the culture media used in the study was performed at the media preparation stage and included the culture medium name, brand, colour, gross appearance, texture and quantity of the weighed powdered media.This was followed by measuring the amount of water to be used and the pH of the broth.After media preparation, QC was done for the performance of the media where the physical appearance (e.g.wrinkling and the presence of precipitates), sterility and the use of E. coli ATCC 25922 strains for antimicrobial susceptibility testing were done.The internal QC was conducted following the CLSI 2020 guidelines. 79

E. coli isolation and identification
All clinical and environmental samples were cultured on MacConkey agar at 37°C for 24 h.The identification of E. coli was determined based on its colony morphology, pigment production and Gram-staining reaction followed by confirmation of isolates using biochemical assays.Therefore, E. coli was identified when the isolate tested positive for indole and lysine but negative for citrate and urea.Additionally, positive E. coli isolates produced a gas, fermented mannitol, and were motile.Further bacterial identification, antibiotic susceptibility testing (AST) and phenotypic detection of ESBL production were conducted using VITEK 2 Compact according to the manufacturer's instructions.

Identification of E. coli isolates by VITEK 2
We used the VITEK ® 2 Compact system (bioMérieux, Marcy-l'Etoile, France) to identify the microorganisms and their susceptibility to antimicrobial agents by examining the biochemical reactions present on microbial identification cards using advanced colorimetry technology.The system's internal optics can read these cards after inoculating them with an unknown organism.Comparing the results to known reactions in the VITEK 2 database enables the system to identify the organism.The system employs a transmittance optical system to interpret the test reactions using different wavelengths within the visible spectrum.During incubation, each reaction is measured every 15 min for turbidity Kasanga et al.  or coloured substrate metabolic products, and an algorithm is employed to remove false readings caused by small bubbles.
The bacterial identification was carried out using an ID-GN card for Gram-negative bacteria (bioMérieux, Marcy-l'Etoile, France).The card is used for automated identification and is designed to identify 135 types of Gram-negative bacilli that are important for fermenting and non-fermenting.To create a bacterial suspension, 3.0 mL of sterile saline was transferred into two clear plastic test tubes, and morphologically similar colonies (pure culture) were added to the first tube using a sterile loop.The density of the suspension with the equivalent standard of 0.50 to 0.63 McFarland was then measured using a turbidity meter (DensiChek), and a 145 μL portion of the suspension was transferred to a second tube.The first vial of bacterial suspension was used for identification, whereas the second was used for AST and ESBL testing.

Antimicrobial susceptibility testing
AST was done using the AST-GN86 card (bioMérieux SA, France).This test provides an efficient solution for both AST and ESBL tests.A comprehensive susceptibility test for 19 antibiotics from different classes, including penicillins, β-lactamase inhibitor combinations, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, tetracycline, nitrofurantoin and trimethoprim/sulfamethoxazole, was conducted using the AST-GN86 card in conjunction with the VITEK 2 system.The results of the susceptibility test were interpreted as sensitive, intermediate or resistant according to the CLSI 2020 guidelines. 79Furthermore, the VITEK 2 system determines the MIC for ultimate accuracy.MDR is defined as non-susceptibility to at least one agent in three or more antimicrobial categories.A confirmatory test called the VITEK 2 ESBL test is used to find out whether E. coli AST-GN86 cards contain ESBLs.In this study, MDR was defined as non-susceptibility to at least one agent in three or more antibiotic classes; XDR was defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories, i.e. bacterial isolates remain susceptible to only one or two antimicrobial categories; Pandrug resistance (PDR) was defined as non-susceptibility to all antibiotics tested. 80

Data analysis
The raw data from the VITEK ® 2 Compact system were entered in Microsoft Excel, cleaned, coded and analysed using Statistical Package for Social Sciences (SPSS) version 25.0 (IBM Corp., Armonk, NY, USA).We used frequencies to describe the distribution of sociodemographic, clinical and environmental variables, and resistance profiles of E. coli to a panel of antibiotics.The prevalence of MDR and ESBL-producing E. coli was determined using descriptive statistics.A P < 0.05 was regarded as statistically significant.
Our study found that the prevalence of E. coli isolates was 47.8% (37.8% clinical and 10.0% environmental).The prevalence of E. coli clinical isolates in our study was lower than the 58.9% reported in the Democratic Republic of Congo (DRC). 81Further, the prevalence of E. coli isolates from environmental sources in our study was lower than that found from the watershed and food-producing animals in the USA (99.3%) 82 and in China (64.7%). 83Also, our study found a lower prevalence of E. coli isolated from environmental sources compared with those reported in Nigeria and Ghana, where the E. coli prevalence was 48.9% from raw milk and 49.5% from drinking water sources. 84,85We reported a higher prevalence of E. coli clinical isolates compared with the 30.3% reported in Palestine 86 and 14.2% in Ethiopia. 87These variations could be partly due to geographical variations, technical differences, the methods used and the experience of the personnel who conducted the laboratory work.
][90][91][92] Penicillins are widely accessed without prescription and are usually inappropriately used, which might contribute to the observed resistance to these drugs. 93The resistance of E. coli to penicillins could also be facilitated by the presence of AmpC β-lactamases encoded by the chromosome of E. coli. 94Additionally, our study revealed that the E. coli isolates were highly resistant to sulfamethoxazole/trimethoprim.6][97] The overuse and misuse of sulfamethoxazole/trimethoprim have contributed to the resistance of E. coli to this drug combination.However, the resistance of E. coli to sulfamethoxazole/trimethoprim has been reported even in individuals who have never used the drug combination. 98ur study also revealed high resistance of E. coli to ciprofloxacin and levofloxacin.4][105][106] The resistance of E. coli to quinolone antibiotics could be due to the occurrence of chromosomal mutations or plasmid-mediated quinolone resistance. 107urthermore, E. coli may also harbour chromosomally encoded AmpC β-lactamases that are capable of hydrolysing cephalosporins, especially when overexpressed. 94Unfortunately, even after a reduction in prescriptions of fluoroquinolones like ciprofloxacin, there appears to be an increase in ciprofloxacin-resistant E. coli. 101he current study also found that most E. coli isolates were resistant to cephalosporins, including cephazolin, ceftriaxone, cefuroxime and cefepime.In Zambia, increased resistance of E. coli clinical isolates to ceftriaxone was reported in previous studies and was revealed to be due to high use of this drug in healthcare facilities. 108,109Two studies conducted in Iraq reported high resistance of E. coli to ceftriaxone and cefotaxime. 89,90A study in the DRC found high resistance of E. coli to ceftazidime. 110][116][117] The present study found that E. coli was highly susceptible to amikacin, meropenem, imipenem, ertapenem, nitrofurantoin, gentamicin and ceftolozane/tazobactam.Our findings are in line with those reported in other studies where E. coli was Antimicrobial resistance profiles of Escherichia coli 90%-100% susceptible to amikacin. 118Additionally, the high susceptibility of E. coli isolates to carbapenems has been reported in other studies. 89,119Intriguingly, the high susceptibility of E. coli to nitrofurantoin and gentamicin has also been reported by other researchers. 89A 100% susceptibility of E. coli to nitrofurantoin was reported in another study. 95These findings are encouraging because nitrofurantoin is used in many countries as a first-line and empirical treatment for urinary tract infections. 95 high prevalence of MDR E. coli (66.5%) was reported in the current study.A comparable but slightly lower prevalence (64.9%) of MDR E. coli was reported in Nepal, 120 with a lower rate (48.7%) reported in Ghana. 121However, a higher prevalence of MDR E. coli was previously reported in Sudan (92.2%), 122 Zimbabwe (84%) 92 and Nigeria (79.2%). 123MDR E. coli is a public health concern and has been associated with increased morbidity and mortality globally. 124This is because MDR infections limit the choice of antimicrobial therapy and make the treatment of infections difficult or impossible. 125,126The high prevalence of MDR E. coli reported in our study and similar studies indicates the extent of this problem globally.Addressing this problem requires a multifaceted holistic approach.
The present study found that 42.8% of E. coli were ESBL-producing.Their occurrence in this study indicates the high resistance to antibiotics including penicillins, third-generation cephalosporins (ceftriaxone, cefotaxime and ceftazidime) and aztreonam, similar to reports from other studies. 127,128This is because ESBLs hydrolyse these antibiotics leading to treatment failure. 111,112Comparable to our findings, a Chinese study reported that 45.7% of E. coli isolated from blood and respiratory samples were ESBL-producing. 118Lower prevalences of ESBL-producing E. coli were reported in Nigeria (16.7%) 123 and in Nepal (38.9%), 120 whereas higher rates were found in previous studies, including 59% in the Central African Republic 129 and 91% in the USA. 130The presence of ESBL-producers is of clinical significance and if not addressed may complicate treatment leading to treatment failure and eventually increased mortality. 131,132ur study highlights the high AMR of E. coli to some of the antibiotics commonly used in humans and the environment.Therefore, the study findings demonstrate the need for heightened surveillance of AMR in humans and the environment.Additionally, our findings demonstrate the need for heightened AMS programmes in hospitals and communities.

Conclusions
This study found that E. coli was highly resistant to some common antibiotics used in humans and the environment.Interestingly, some isolates were highly susceptible to some priority antibiotics used in humans.However, the high prevalence of MDR and ESBL-producing E. coli in this study is of public health concern and can affect the treatment of infections.There is a need to heighten the surveillance of AMR in humans and the environment.Further, AMS programmes should be instigated and strengthened in healthcare facilities and in communities to reduce the emergence of AMR and prevent its potentially fatal consequences.

Figure 1 .
Figure 1.Townships sampled in the Lusaka district.